J Endocrinol Invest (2014) 37:1009–1014 DOI 10.1007/s40618-014-0166-6

ORIGINAL ARTICLE

BRAF mutation analysis in thyroid nodules with indeterminate cytology: our experience on surgical management of patients with thyroid nodules from an area of borderline iodine deficiency P. Agretti · F. Niccolai · T. Rago · G. De Marco · A. Molinaro · M. Scutari · C. Di Cosmo · G. Di Coscio · M. Vitale · M. Maccheroni · P. Vitti · M. Tonacchera 

Received: 4 July 2014 / Accepted: 19 August 2014 / Published online: 7 September 2014 © Italian Society of Endocrinology (SIE) 2014

Abstract  Purpose  Fine-needle aspiration (FNA) with cytologic evaluation is the most reliable tool for malignancy prediction in thyroid nodules, but cytologic diagnosis remains indeterminate for 12–18 % of nodules. BRAF V600E mutation has been reported to show a high specificity for malignant thyroid nodules and the use of this marker to refine indeterminate FNA cytology results may be a useful diagnostic adjunctive tool in the pre-operative evaluation of thyroid nodules. The aim of this study was to estimate the prevalence of BRAF exon 15 mutation (V600E) and its clinical value as a diagnostic tool in a series of thyroid nodules with indeterminate cytology from an area of borderline iodine deficiency. Subjects and methods  One hundred and fifty-three thyroid samples obtained by FNA of thyroid nodules from 151 patients were subjected to the analysis of BRAF V600E mutation by direct sequencing. In the study 54 nodules with indeterminate cytology, 56 benign and 43 malignant thyroid nodules were included.

P. Agretti · F. Niccolai · T. Rago · G. De Marco · A. Molinaro · M. Scutari · C. Di Cosmo · M. Maccheroni · P. Vitti · M. Tonacchera (*)  Department of Clinical and Experimental Medicine, Section of Endocrinology, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124 Pisa, Italy e-mail: [email protected] G. Di Coscio  Department of Oncology, Section of Cytopathology, University of Pisa and University Hospital of Pisa, Pisa, Italy M. Vitale  Department of Medicine and Surgery, University of Salerno, Salerno, Italy

Results  V600E BRAF gene mutation was demonstrated in 19/43 malignant nodules, in 0/56 benign nodules and in only 1/54 indeterminate nodules that, after histology, turned out to be at a papillary thyroid carcinoma. Conclusions  The application of BRAF exon 15 analysis showed limitations when applied to discriminate thyroid nodules with indeterminate cytology if wild-type BRAF is found, and there is no role for avoiding diagnostic thyroid surgery. Keywords  Thyroid nodules · Fine-needle aspiration · Follicular proliferation · Genetic analysis

Introduction Thyroid nodules are the most common thyroid disease, with an incidence of 1–9 % in iodine sufficient areas that markedly increases in iodine deficient countries. The majority of thyroid nodules are benign and the incidence of thyroid cancer is low, accounting for about 5 % of nodules [1, 2]. Papillary thyroid carcinoma (PTC) is the most frequent thyroid cancer, accounting for about 85–90 % of all thyroid cancers, whereas follicular thyroid carcinoma (FTC) accounts for about 10 % or less and anaplastic carcinomas are very rare [3–5]. Fine-needle aspiration cytology (FNAC) of thyroid nodules is the most specific and most sensitive method to differentiate malignant from benign thyroid nodules [6–10]. However, in the case of follicular lesions, cytology is unable to discriminate between benign follicular adenoma and thyroid carcinoma and whether the carcinoma is follicular or the follicular variant of papillary thyroid carcinoma [11–15]. This diagnosis of indeterminate FNAC, also named follicular lesion of undetermined significance/atypia of undetermined significance (FLUS/AUS)

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[4, 16–18] occurs in 12–18 % of all cases and is due to the fact that cannot be detected by cytology the diagnostic criteria of FTC. Therefore, patients with this diagnosis have to be subjected to diagnostic thyroid surgery, resulting in a malignancy in 5–30 % of cases [4, 16]. In addition, according to the recent SIAPEC-AIT guidelines [18], indeterminate nodules should be distinguished in TIR3A and TIR3B, and only TIR3B is recommended to undergo diagnostic thyroidectomy. TIR3A nodules should represent up to 50 % of all indeterminate nodules and, therefore, TIR3B should be comprised between 6 and 9 % of all nodules. The molecular analysis on cytological specimen is nowadays feasible and, when including a panel of complete proto-oncogenes, increases the diagnostic accuracy of traditional cytology [19]. PTC is characterized at the molecular level by activating mutations or chromosomal rearrangements of BRAF, RET, RAS, or TRK genes which can be detected in about 70 % of PTC. FTC is characterized by activating RAS mutations and PAX8-PPARγ rearrangements which are identified in up to 63% of FTC [19–25]. Because specific somatic mutations are unknown in >30 % of the FTC and are merely unknown for follicular adenomas or hyperplastic nodules [19], a diagnostic discrimination of FTC and benign follicular thyroid nodules with indeterminate cytology by molecular methods has only been reported in very few cases and, therefore, remains a major diagnostic challenge with a large potential impact on treatment decisions and treatment costs. Just think that about 70–80 % of thyroid lobectomies performed solely for diagnostic purposes are benign [26]. BRAF V600E amino acid substitution is the most common genetic alteration in PTC and is identified in 40–45 % of these tumors [22]. BRAF gene codifies for a serine/ threonine-protein kinase involved in sending signals inside cells, which are involved in directing cell growth. This protein is a key molecule in the mitogen-activated protein kinase pathway (MAPK) and is mutated in many human malignancies although the frequency varies significantly between different types of cancer [27]. Over 30 mutations of BRAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. In particular, V600E BRAF mutation accounts for 90 % of BRAF mutations in human cancers and possesses the hallmarks of a conventional oncogene [28]. This residue is one of those that contribute to stabilization of an inactive conformation of the BRAF kinase domain. Mutations of these residues destabilize this inactive conformation, promoting the active state [28]. For this reason, the analysis of this mutation represents a useful and accurate pre-operative diagnostic tool to FNAC providing additional information in the differential diagnosis of malignant thyroid nodules showing indeterminate cytology results [22].

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J Endocrinol Invest (2014) 37:1009–1014

The aim of this study was to estimate the prevalence of BRAF V600E mutation and to evaluate its clinical value as a diagnostic tool in a series of thyroid nodules with indeterminate cytology from an area of borderline iodine deficiency.

Subjects and methods Patients and FNA analysis Ultrasound-guided fine-needle aspiration cytology was performed as a part of the standard diagnostic protocol for patients with thyroid nodules at Endocrinology Section of the University Hospital of Pisa, Italy [8]. Great care was used to collect material only from nodular lesions with the help of ultrasound. One hundred and fifty-three thyroid samples obtained from 151 consecutive patients (112 females of medium age 45.4 ± 11.7 and 41 males of medium age 48.0 ± 8.6 years) were collected and included in the study (we selected the first 56 benign nodules, the first 43 malignant nodules and the first 54 indeterminate nodules). All the patients came from a region of Italy with borderline iodine deficiency (Tuscany). After the aspirate was smeared for conventional cytology, the leftover material in the needle was dispersed in TRIZOL reagent for genomic DNA isolation and molecular analysis. A specimen was considered as satisfactory if there were six groups of epithelial cells with at least ten cells per group [29]. According to fine-needle aspiration cytological analysis, the nodules were classified as benign, indeterminate, suspicious for malignancy or malignant, and non-diagnostic or inadequate (due to limited cellularity or poor preservation and fixation) following the guidelines of National Cancer Institute Thyroid Fine-Needle Aspiration State of the Science Conference [16, 17]. In this series, nodules with inadequate cytology were not considered for further molecular analysis. Laboratory evaluation of thyroid function Serum-free thyroxin (FT4) and free triiodothyronine (FT3) were measured with a chemiluminescent method (Vitro System, Ortho-Clinical Diagnostics, Rochester, NY, USA). Thyrotropin (TSH) and calcitonin were assessed by ultrasensitive commercial chemiluminescent method (Immulite 2,000; Diagnostic Products, Los Angeles, CA, USA). TPO and Tg antibodies were measured using a two-step immunoenzymatic assay (AIA-Pack TgAb and TPOAb; Tosoh, Tokyo, Japan). Genomic DNA isolation Cells from FNA samples were collected in TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA, USA)

J Endocrinol Invest (2014) 37:1009–1014 Fig. 1  Cytology, genetic analysis of BRAF V600E mutation and histology of 153 thyroid nodules included in the study

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CYTOLOGIC RESULT

Benign (n=56)

GENETIC ANALYSIS

wild-type BRAF (n=56)

HISTOLOGIC RESULT

Not available (n=56)

V600E BRAF (n=19)

PTC (n=19)

wild-type BRAF (n=24)

PTC (n=24)

V600E BRAF (n=1)

PTC (n=1)

Malignant (n=43)

FLUS (n=54) wild-type BRAF (n=53)

Benign lesion (n=40) PTC (n=12) FTC (n=2)

and genomic DNA isolation, in addition to total RNA extraction for different purpose, was performed according to the manufacturer’s instructions. Genetic analysis All DNA samples were analyzed for V600E BRAF mutation by PCR amplification and direct sequencing. A fragment of the exon 15 of BRAF gene was amplified by PCR using specific primers and, after purification, was directly sequenced using BigDye Terminator Kit and 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) as previously described [30].

Results Patients management: thyroid function, cytology and histology A total of 151 patients, to which belonged the 153 samples, entered the study. We obtained 56 benign thyroid nodules, 43 malignant thyroid nodules and 54 thyroid nodules with indeterminate cytology (hereafter FLUS) on the basis of the cytological response. Fifty-six benign thyroid nodules belonged to 55 patients, 14 males with a medium age of 47.1 ± 10.1 years, and 41 females with a medium age of 47.3 ± 11.9 years. Forty-three malignant thyroid

nodules belonged to 42 patients, 16 males with a medium age of 43.4 ± 15.1 years and 26 females with a medium age of 44.8 ± 12.0 years. Fifty-four FLUS belonged to 54 patients, 13 males with a medium age of 52.5 ± 9.4 years and 41 females with a medium age of 43.9 ± 11.3 years. Serum FT4, FT3 and TSH values were in the normal range in all patients. No serum anti-thyroglobuline and antithyroperoxidase antibodies were detectable. Serum calcitonin was undetectable in all patients. All patients with benign thyroid nodules were followed conservatively for at least 5 years by annual ultrasound examination, while all patients with malignant or indeterminate cytology underwent thyroid surgery soon after completion of the clinical and cytological evaluation. All nodules with an FNA indicative of malignancy were PTC at histological examination and of the 54 FLUS, 12 were PTC, 2 FTC and 40 benign lesions at histological examination. Besides, of the 12 papillary carcinomas, three were microcarcinomas